Kinetics of Hydrogen Peroxide Production by Lactobacilli

3.3.1. Inoculum Buildup for the Growth Experiments

1. Lactobacilli subcultivation: L. crispatus CRL 1266, L. paracasei CRL 1251 and 1289 (from human vagina), and L. acidophilus 1012 and 2014 (from bovine vagina). These Lactobacillus strains were selected for hydrogen peroxide production and inhibition of pathogenic microorganisms, according to the screening performed. The lactobacilli were grown as described in Subheading 3.1.2., step 1.

2. The third culture was centrifuged (10 min, 2000g), and the spent supernatant was discarded. The pellet was washed with 3 mL saline solution and then resuspended in the same solution to reach a final OD of 1.4 at 540 nm. This bacterial cell suspension was used as the inoculum for the growth experiments.

3.3.2. Kinetics of Hydrogen Peroxide Production

1. The lactobacilli were inoculated (2% v/v) into 100 mL of each medium (250-mL Erlenmeyer flasks) and then incubated in a water bath with and without agitation, at constant temperatures (30, 37, and 44°C). Samples were withdrawn at different times from lactobacilli culture (every 2 or 3 h) to perform the analytical procedures (see Note 18).

2. Optical density measurements (OD at 540 nm): The initial turbidity of the growth media was corrected to absorbance value 0 (zero) with the corresponding blanks of culture media placed in glass cuvets.

3. The samples of lactobacilli cultures were placed in small plastic bottles to determine the pH with a digital pHmeter, previously calibrated with the reference standard buffers.

4. Determination of colony forming units (CFUs): The samples were serially diluted (10-fold) in peptone water, from 1:10 to 1:1,000,000. Aliquots of each dilution were placed on the plates, and 12-15 mL of 45°C melted culture medium (LAPTg or MRS agar, depending on whether the previous lactobacilli growth was in LAPTg or MRS broth) were added. The plates were incubated at 37°C for 48 h (see Note 19).

5. Quantification of hydrogen peroxide: Samples taken at different times of lactobacilli culture were centrifuged at 2000g for 10 min, and the supernatant fluids were used to perform the assay (spectrophotometric method modified by using o-dianisidine horseradish peroxidase), as described in Subheading 3.2.2.3. (see Note 20).

4. Notes

1. To enhance the growth of Corynebacterium, BHI medium can be enriched with: 1:10 v/v hemine solution (hemine 50 mg, 1 mL 1 N NaOH, in 100 mL of solution) and 0.02:100 v/v vitamin K.

2. Before performing the plate diffusion technique, is necessary to set up a calibration curve of optical density vs CFU/mL, to select the best inoculum of pathogen microorganisms to achieve 107-108 CFU/mL in the plates. In the case of Corynebacterium pyogenes, the bacterial suspension (OD540 = 0.24) was made from the microorganisms grown on the surface of the agar plates and then diluted 1:10.

3. Caution: TMB is irritating to the eyes, respiratory system, and skin, there is a possible risk of irreversible possibly mutagenic, effects.

4. Caution: Methanol is flammable and toxic. Avoid contact with the skin, and store far from flames or spark sources.

5. Solutions A and B were prepared immediately before the TMB agar plate preparation.

6. The TMB agar plates can be stored at 2-8°C for 1 wk maximum.

7. When performing these assay, an appropriate dilution of lactobacilli must be spread over the surface of TMB agar plates to obtain isolated small numbers of colonies. Is possible to use a surface dissemination method with a loop to obtain single colonies at the end of the dissemination.

8. The exposure of the plates to air is a very important step to allow rapid maximum color development of the colonies over the TMB agar plates.

9. The colonies that turn blue after exposure to air and TMB agar are those microorganisms that produce higher amounts of H2O2 when they grow in liquid media.

10. Caution: o-dianisidine is toxic and may cause cancer or heritable gene damage. It is irritating to the eyes, respiratory system, and skin; wear suitable protective clothing, gloves, and eye/face protection, and do not breathe the dust.

11. Solutions C and D were prepared immediately before the reagent solution preparation.

12. The final concentration of peroxidase in the reagent solution, modified from the original technique (19), was adjusted to 1.8 ^g/mL.

13. The reagent solution should be stored in a dark flask at 4°C for 2 mo maximum.

14. Caution: 30% H2O2 is corrosive and flammable; avoid contact with the skin, and wear suitable protective clothing, gloves, and eye/face protection.

15. The solutions containing different concentrations of H2O2 were prepared on the day on which the standard curve was performed, because storage of H2O2 can result in complete loss after a short time.

16. The third lactobacilli culture, for the screening performed with the spectrophotometric method modified by using o-dianisidine horseradish peroxidase, must always be grown with shaking, because there are no detectable levels of H2O2 in nonagitated cultures.

17. To determine the H2O2 produced during the growth of microorganisms, the reference solution and the solutions with different H2O2 concentrations used to perform the standard curve were prepared with the corresponding sterilized growth medium (LAPTg or MRS broth). If the OD of the assay mixture is out of the linear range of the standard curve, the samples should be diluted with the growth medium to perform the assay again, and the H2O2 concentration values obtained should be multiplied by the dilution factor.

18. The presence of oxygen in the environment of lactobacilli growth can influence their physiology. In fact, some lactic acid bacteria possess oxidases that reduce oxygen to hydrogen peroxide, oxidizing substrates such pyruvate or NADH (25). The H2O2-pro-ducing lactobacilli were grown in agitated and nonagitated cultures in order to determine the factors that affect the production of this metabolite and the most favorable conditions to obtain the highest biomass. The vigorous shaking provided permanent aeration to cultures, whereas no shaking was used for nonaerated cultures. The results obtained showed that O2 must be supplied through agitation to detect the H2O2 in spent culture supernatant fluids, whereas there was no detectable production (within the sensitivity range of the assay) of the metabolite in nonagitated cultures.

19. In aerated conditions, it is recommended to spread an aliquot of undiluted lactobacilli culture on the surface of agar plates because the growth of microorganisms can be inhibited by the H2O2 produced. The toxicity of H2O2 is caused by an effect exerted by the molecule itself or by superoxide anion and hydroxyl radicals, which cause oxidative stress in the cells, decreasing the number of viable cells (25).

20. The samples must be withdrawn from the flasks every 2-3 h to minimize the time in which the flasks are outside the incubator. The supernatants separated by centrifugation can be stored at 4°C for 2-3 h up to the H2O2 determination in order to avoid the loss of this metabolite.

Acknowledgments

This paper was supported with grants from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina), PIP 359, and Beca Ramón Carrillo-Arturo Oñativia para Investigadores formados (Ministerio de Salud Pública de Argentina).

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